MUC1 is a glycoprotein highly expressed in a number of human epithelial malignancies, including breast, prostate, colon, ovarian and pancreatic carcinomas, as well as on the malignant plasma cells of multiple myeloma. Although alternative splicing can generate a variety of MUC1 isoforms, the most intensively studied MUC1 protein is a type I transmembrane protein comprised of a heavily glycosylated extracellular domain containing a tandem-repeat-array, a transmembrane domain, and a cytoplasmic domain (MUC1/TM). MUC1/TM is proteolytically cleaved soon after its synthesis generating two subunits, α and β which specifically recognize and bind in a strong non-covalent interaction (see, FIG. 1, MUC1/TM). Cleavage of MUC1 into the two subunits occurs in the SEA module, a highly-conserved domain found in a number of cell-tethered mucin-like proteins (Levitin, et al., J Biol Chem (2005) 280:33374-86). Shedding of the α-subunit from the cell membrane results in soluble tandem-repeat-array-containing MUC1 in the peripheral circulation, and it is this molecule which is used to determine serum MUC1 levels in patients with MUC1 positive malignancies.
The presence of the soluble α-subunit MUC1 protein in the circulation presents a singular difficulty in delivering adequate amounts of anti-MUC1 antibodies to directly target MUC1-expressing malignant cells. This is because the most immunogenic part of MUC1 is the tandem-repeat-array and almost all anti-MUC1 antibodies generated to date exclusively recognize epitopes in that immunogenic region. Sequestration of anti-tandem repeat antibodies by the soluble, circulating MUC1 α-subunit severely limits the amount of antibody which can successfully bind MUC1 on the cell surface. Furthermore, deposition of immune complexes of anti-tandem repeat antibodies and its soluble, circulating MUC1 target can lead to significant end-organ damage.
In recent years, numerous efforts have been made to generate effective anti-MUC1 antibodies using the full-length MUC1/TM molecule as immunogen. The major obstacle hampering those attempts is that immunization with the whole MUC1/TM molecule invariably results in an antibody response composed almost in its entirety of antibodies recognizing epitopes on the highly immunogenic tandem repeat array. For ultimate application in in vivo targeting of MUC1-expressing tumor cells, such antibodies pose all the shortcomings inherent in anti-repeat antibodies, as detailed above.
Antibodies recognizing MUC1 epitopes tethered to the cell surface potentially obviate these difficulties. Although conceptually simple, generation of monoclonal antibodies to MUC1 stably tethered to the cell surface first requires characterization of cell-bound, non-shedding epitopes. The junction formed by the MUC1 α-subunit binding the membrane-tethered β-subunit provides such an epitope.